Alternative splicing modulation as a therapeutic tool

(1)

Alternative splicing modulation

as a therapeutic tool

2019-04-30

Jordi Hernández Ribera

(2)

Transcription

Pre-messenger

RNA

Gene expression

DNA

jordi.hernandez@crg.eu

(3)

CH2 O PO2- O 5´ O OH 2´ Intron 5’SS BP 3’SS A PYT

Exon A GU YYYY AG Exon B

RNA splicing

(4)

5’SS 3’SS A

Exon A 3’OH YYYY AG Exon B

Intron

RNA splicing

(5)

Exon A Exon B

Lariat Spliced Product

RNA splicing

A

(6)

The splicing reaction

(7)

Cis-acting

sequences sequencesCis-acting sequencesCis-acting

Trans-acting

factors Trans-acting factors

Alternative splicing regulation through cis/trans-acting elements

(8)

The combinatorial effects of splicing factors will determine the splicing outcome

(9)

Dvinge et al., 2016

Different types of alternative splicing events

(10)

Alterations in RNA splicing and cancer

Modified from Hanahan et al. 20011 hTERT Δ4EGFR NUMB Cyclin D1b Δ Np53 Bcl-xl Soluble Fas VEGFxxx E-cadherin_CD44 PKM2 LDHC HLA-G1 MHC1 Δ Np53 Rac1b STAT3 jordi.hernandez@crg.eu

(11)

8 9 10

NUMB

Isoform

p71

Misquitta-Ali CM, et al. Mol Cell Biology. 2011 Bechara E, et al. Molecular Cell. 2013

Hernández J, et al. RNA Biology. 2016

Cell proliferation is controlled

by

NUMB alternative splicing

8

9

10

8 10

Proliferative

Antiproliferative

Isoform

p65

(12)

0.0 0.2 0.4 0.6 Breast Invasive Carcinoma

Head and Neck Squamous Cell Carcinoma Kidney Chromophobe Kidney Renal Papillary Cell Carcinoma Liver Hepatocellular Carcinoma Lung Adenocarcinoma Lung Squamous Cell Carcinoma Prostate Adenocarcinoma Thyroid Carcinoma Tissue Type normal tumor Colon Adenocarcinoma Kidney Renal Clear Cell Carcinoma PSI

Figure 1

* * * * * * Caterina Coli

Eduardo Eyras Data from The Cancer Genome Research Atlas

Elevated NUMB inclusion in tumor samples

n=57

(13)

KRAS

EGFR

Unknown ALK

MET

Alterations in lung adenocarcinoma

Chan BA, Hughes BG

Transl Lung Cancer Res; 2015

Lung Cancer

Rest of Cancers Prostate Cancer Breast Cancer Colon Cancer

The oncological unmet need: lung cancer

1.8 million diagnoses per year and 1.6 million death per year

(14)

KRAS

EGFR

Unknown

ALK

MET

Alterations in lung adenocarcinoma

Chan BA, Hughes BG

Transl Lung Cancer Res; 2015

Lung Cancer Rest of Cancers Prostate Cancer Breast Cancer Colon Cancer

Increased levels of

NUMB exon 9

inclusion

The oncological unmet need: lung cancer

1.8 million diagnoses per year and 1.6 million death per year

(15)

Patient survival predicted by NUMB inclusion

Time in days Su rv iv al p ro p or ti on

NUMB skipped exon (chr14: 73749067-73744001, negative

strand, hg19)

PSI cutoff: 0.65; Log-rank p-value: 2.46e-05

Inclusion levels >= 0.65 Inclusion levels < 0.65

0 1k 2k 3k 4k 5k 6k 7k 0 0.25 0.5 0.75 1

Log rank p-value 2.46-6

Javier Tapial

Days

Su

rv

iv

al

p

ro

po

rt

io

n

Patients with NUMB PSI > 0.65 Patients with NUMB PSI < 0.65

n=95 n=408

(16)

Three key regulatory regions were identified

NUMB exon 9

AON 1-2 AON 2-3 AON 3-4 AON 4-5 AON 5-6 AON 6-7

3’ SS 5’ SS

AON 1 AON 2 AON 3 AON 4 AON 5 AON 6 AON 7

BP RND

A

CNT

HEK293-RG6NUMB AON 3-4 AON 4-5 AON 5-6

0.27 ± 0.06 ± 00 AON 1 0 ± 0 PSI RND 0.93 ± 0.04 AON 5-6 AON 6-7 5’ SS 0.18 ± 0.07 ± 0.020.05

AON1 AON2 AON3

CNT

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CNIO 201611 0 15 30 45 60 0 5 10 15 CNT AON1 Days Fol d C ha nge Tu m or V ol um e CNIO 201611 0 15 30 45 60 0 5 10 15 CNT AON1 Days Fo ld C h an g e T u m o r V o lu m e K-rasLSLG12V induction 4OH-TMX RERTert_, K-rasLSLG12V 30 0 15 Tumour detection 4-5 months 45 Days

Dose Dose Dose Dose Dose Dose Dose

60

Dose Dose

Weekly administration of AON1 reduces tumour growth

6! Miguel Rovira

Manuel Serrano

Technology!

Lung Cancer Model

Tumor initiation CT CNIO 201611 0 15 30 45 60 0 5 10 15 CNT AON1 Days Fo ld C h an g e T u m o r V o lu m e Control

*!

AON1 reduces tumor growth in genetic K-Ras driven mouse model of LUAD

Fo ld C ha ng e Tu m or V ol um e Weeks CNT AON1 0 2 4 6 8 Miguel Rovira Manuel Serrano n=6 n=7 p-value 0.0397

*

(18)

Orthotopic A549 all AON

0 1 2 3 4 5 6 7 8 0 50 100 150 200

Weeks

Fo

ld

C

h

a

n

g

e

Tu

m

o

r V

o

lu

m

e

CNT

AON1

AON6-7

AON1 administered intranasally modulates NUMB alternative splicing in tumours

Pablo J. Fernández Manuel Serrano RN D AON 1 0.00 0.01 0.02 0.03 PSI

A

*** Salin e RND AON 1 0.00 0.05 0.10 0.15 PSI

B

* *

C

Figure 5

1 ₁₅ ₃₀ ₄₅ ₆₀ 0 1 2 3 5 10 15 Days T u m o r V o lu m e m m K-rasLSLG12V induction 4OH-TMX RERTert_, K-rasLSLG12V 30 0 Sacrifice Treatment 15 Tumour detection 4-5 months 45 Days CT

Implantation of A549 cells

Mesurement Tumor detection

AON1 AON1 AON1 AON1

1 week 1 week 1 week 1 week Total 2 months

AON1 Weeks CNT AON1 AON3 Jessica Vitos Anna Ribo p-value 0.0078

**

n= 15 n= 11 n= 11

Anti-tumoral effect of AON1 in vivo orthotopic LUAD model

Fo ld C ha ng e Tu m or V ol um e jordi.hernandez@crg.eu

(19)

AON1 reduces the colony formation capacity of several cell lines

RND AON1 A549 H1395 H1666 H1975 96 KLC197 KLC2 MCGL KLC3LS KLC4

H226 MCF7 T47D LS147 HT29 pc3 _{DU145 QGP1 Capan2} _{HeLa Saos2 SK4SN Huh7 FTO-2B HepG2}

0

50

100

150

200 Baseline-corrected of Clonogenics

Cell Type

Re

la

tiv

e

Nu

m

b

e

r o

f Co

lo

n

ie

s

LUAD human mice LSCC _Breast _Colon Prosta te Panc

reatic Cervi x Osteo sarco ma Neurob lastom a Hepato carci noma *** *** *** * *** *** ** * *** *** * ** ** * *** jordi.hernandez@crg.eu

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Conclusions

• Lung cancer is an unmet medical need.

• NUMB alternative splicing represents a novel target, altered in all LUAD samples.

• NUMB alternative splicing modulation represents a novel approach, validated in vivo, for lung cancer treatment.

85% mortality rate

Lung Adenocarcinoma Ex on In cl usi on 0.6 0.4 0.2 0

1.8 million diagnoses per year

1.6 million death per year

(21)